Precipitation is a widely used method for the recovery of proteins from various solutions. Proteins in solution vary greatly in their solubility depending on their amino acid composition and the characteristics of the solvent (such as ionic strength and temperature). A number of methods for precipitation of proteins are well known. Protein crystallization, for example, is frequently used to precipitate proteins, but this method requires typically comprehensive development to achieve right crystallization conditions.
Isoprecipitation is another frequently used protein precipitation method, making use of the fact that a proteins solubility has a minimum at the protein's isoelectric point (PI). However, due to posttranslational modifications a number of proteins do not have a well defined PI. For example, coagulation factor VII (“FVII” or “FVIIa” (when referring to the activated form of FVII)) comprises y-carboxy groups and sialic acid groups, but not in any fixed number, making the pI of FVII difficult to define.
Protein precipitation also frequently is induced by addition of an organic solvent, to alter the nature of the solution in which the protein is contained. Traditionally, it has been believed that if there is a significant decrease in the dielectric constant of the medium with the addition of an organic solvent, the solubility of any proteins in a solution should also decrease and proteins should precipitate. More recent research has suggested that dehydration of protein surfaces is a reason (if not the reason) for precipitation of proteins in the presence of organic solvents.
“Salting out” protein precipitation methods have been used extensively, particularly before the development of more recently developed chromatographic purification methods. Salting out of proteins makes use of the fact that a protein's solubility often decreases at high salt concentrations (and proteins can therefore be made to precipitate under such conditions). Various salts have different salting out effects, but the general principles for salting out are presented in the so-called Hofmeister series that have been known for more than 100 years. Besides the particular salt used, the nature of the protein in question also influences its ability to be precipitated by such a method. Ammonium sulphate is among the salts that most often has been used for precipitation of proteins.
The methods of salting out precipitation, organic solvent precipitation, and isoprecipitation, have frequently been used in isolation, rather than in combination with each other.
One method that makes use of a high salt concentration (typically using ammonium sulphate in an order 0.3-5 M) in combination with a water soluble organic solvent (typically t-butanol) is described in the so-called Three Phase Partitioning (TPP). t-butanol is regarded as completely miscible with water, but at high salt concentrations a two phase system occurs, the lower phase is the aqueous salt phase and the upper is t-butanol phase. However, if protein is present in such a system, a third phase develops in the boundary between the two above-mentioned phases as precipitated protein. Large amounts of t-butanol (17-33%) or other organic substance are required (relative to the aqueous phase) in such a method (see, e.g., C. Dennison et al., Protein Expression and Purification 11, 149-161 (1997), which discloses a so-called Three Phase Partitioning (TPP) system, a system wherein two liquid phases are formed, and where proteins partion between the liquid phases, resulting in a third solid phase in the boundary between the two liquid phases). The combination ammonium sulphate and t-butanol are far the most used, in such methods, however others salts and other organic compounds (such as n-butanol) have also been tried.
J. Leonil et al., Enzyme Microb. Technol. 16, 591-595 (1994), describes precipitation of peptides from a tryptic digest of casein using NaCl and ammonium sulphate. The authors found that particularly pH influenced precipitation and in particular low pH values were preferable. However, no organic compounds were used in the precipitation method.
Solvent effects on solubility and stability of IGF-1 also have been studied (J. Fransson et al., Pharmaceutical Research 14, 606-612 (1997)). The goal of this work was to investigate circumstances in which a stable protein solution could be obtained without negatively impacting IGF-1's tertiary structure. The report mentions that IGF-1 precipitates in 145 mM NaCl and 140 mM benzyl alcohol (benzyl alcohol is a commonly used preservative known to have a tendency to promote protein precipitation). However, the work of Franson et al. related to formulation and stabilisation of IGF-1, rather than methods for protein precipitation, such that the amount of protein precipitated was small (being an unintended effect).
Su et al, Process Biochemistry 41(2), 257-263 (2006) describes a so-called aqueous two-phase system for separation of proteins, which two-phase system can be brought about by mixing polyethylene glycol and a salt in water and wherein both phases are liquid. Such systems tend to form two liquid phases. If a mixture of proteins are present they will partion unequally between the two aqueous phases, giving rise to a separation of the proteins.
U.S. Pat. No. 5,728,559 discloses the isolation of proteins and in particular enzymes from aqueous solutions by crystallization by addition of a salt at a concentration of 1.5 M or below, but without any pH adjustments, followed by addition of a water soluble polymer resulting in a crystalline product after stirring. The polymer is typically a glycol such as polyethylene glycol.
EP0474391 discloses a method in which an acylated protein (insulin) may be isolated by adjusting the pH to a value near the pI of the protein. Then a suitable amount of an alcohol is added. In the example ethanol is used in an amount of 0.46 liter per liter solution to give a precipitation, in fact a so-called isoprecipitation is described. No salt is added.
EP1561756 discloses the removal of impurities like DNA and viruses from a protein solution (particularly antibodies). This is done by lowering the pH to a value equal to or lower than the pI of the protein with low conductivity. Under these conditions impurities as particles may be removed, while the protein of interest remains in solution. No organic solvent is used.
There remains a need for alternative and improved methods for precipitating proteins. The present invention described herein provides such methods. These and other advantages and features of the invention are further described in the description of the invention provided herein.